This University of Wisconsin Bioengineering Research Partnership proposes to develop technology capable of rapid, inexpensive, and robust production of long dsDNA molecules (genes) up to 10 kilobases in length. The genes will be assembled from individual oligonucleotides synthesized in parallel on DNA arrays using the Maskless Array Synthesizer (MAS), which permits the production of custom DNA microarrays with 786,000 features in only 4 hours. Light-directed "safety-catch" release chemistry will permit desired array components to be released from the surface in sets to form specific sub-assemblies, which in turn will be assembled into the final long dsDNA product. Biological error correction methods, based upon the ability of the mismatch-binding protein MutS to recognize and remove mismatched duplexes, will ensure high fidelity. All of these functions will be merged into an integrated system - the Automated Gene Synthesis (AGS). The ability to make complete genes on demand, inexpensively, and with rapid turn around time, has revolutionary implications for a wide range of biological and medical research. The UW Bioengineering Partnership is made up of an exceptional interdisciplinary team of researchers with expertise in the areas of engineering, chemistry, informatics, and molecular biology and cellular biology. Team members have a proven track record in developing cutting edge tools for biological research. The inclusion of NimbleGen Systems on the team, as an industrial partner, brings additional resources in chemistry and MAS technology to accelerate development. In addition to the primary focus of the group upon total gene synthesis, this bioengineering effort will also apply the massively parallel control of light to fuel basic research in the areas of combinatorial chemistry of small molecules and the detection of surface interactions. Biological relevance of the technologies will be ensured by means of three specific applications: the rapid low cost production of DNA for gene targeting in stem cells (all five of the cell lines to be used in this proposal (HI, H7, H9, H13, and H14) are all listed on the NIH registry), high-throughput protein synthesis, and high density on-chip SNP detection using surface invasive cleavage reactions.